Clathrin-mediated endocytosis (CME) is essential for regulating signaling pathways and internalizing nutrients. During CME initiation, scaffolding proteins that bind to the plasma membrane and adaptor proteins that bind cargo receptors nucleate assembly of clathrin-coated pits (CCP). As the CCP mature, membrane curvature progresses until scission by the GTPase dynamin separates the newly formed vesicle from the plasma membrane. Many curvature effectors have been associated with CME. These proteins are thought to help stabilize membrane curvature as it forms, and promote the membrane deformations necessary for the formation of a vesicle, but their exact role in CME regulation remains elusive. Our preliminary data show that curvature effectors are recruited to areas of artificially induced curvature. Further, live-cell imaging of CME markers revealed that these were active sites of CME. While these findings establish that the number of active CME sites is increased at areas of curvature, the mechanism of this effect is unclear. The objective of the work proposed here is to define the role of membrane curvature in initiation and progression of endocytosis. Specific aims: Regions of mechanically induced curvature are active sites for CME. Whether this increase in CME is due to the ability of the curved membrane to act as a signal of CME site initiation or due to a change sequential recruitment of CME curvature sensors is unknown. Therefore, we propose the following specific aim: to determine the role of membrane curvature in CME initiation and progression. Additionally, aim 2 addresses the role of curvature effector proteins in dynamin fission. Study design. We will analyze the dynamics of CME in cells expressing endogenously-tagged fluorescent CME curvature effector proteins and CME marker proteins on novel nano-fabricated substrates, recently designed by our collaborators. These substrates have nano scale structures that induces curvature of the plasma membrane. To address what role membrane curvature plays in CME initiation and regulation, we will quantify by confocal and super-resolution microscopy the number of initiation events at positions of induced curvature in relation to the planar areas of plasma membrane. In addition, we will define the order of recruitment of key CME proteins to determine if curvature acts as checkpoint to regulate late-stage CCP formation and vesicle scission. We will also determine the role curvature effector domains in dynamin- mediated fission through live cell studies using the nanostructure arrays and apply our findings to reconstitute vesicle fission in a model membrane system composed of unroofed mammalian cells and cytosolic extract derived from genome edited cells.